PB2553: GENOTYPIC CHARACTERIZATION OF RARE INHERITED ANAEMIAS USING NEXT GENERATION SEQUENCING METHODS
Authors: Ganesh Kumar Viswanathan, Jasmita Dass, Rishi Dhawan, Mukul Aggarwal, Pradeep Kumar, Tulika Seth, Manoranjan Mahapatra
Journal: HemaSphere (2023)

Topic: 28. Enzymopathies, membranopathies and other anemias Background: Inherited anaemias have a genetic basis. The diagnosis of this group of disorders includes clinical evaluation and directed laboratory tests. A proportion of cases remain undiagnosed even after extensive laboratory tests; genetic testing by next generation sequencing (NGS) is helpful to identify the underlying genetic cause. This study evaluates the utility of NGS for diagnosis of rare inherited anaemias. NGS testing was done using targeted gene panels to identify the genetic cause of otherwise uncharacterised anaemia. Aims: 1. Identify rare inherited anaemias in patients presenting to the anaemia clinic in the Department of Haematology. 2. Genotypic characterization of rare inherited anaemias using next generation sequencing methods. Methods: Retrospective analysis of inherited anaemia patients presenting to the hematology clinic was performed. Age group of the patients was <40 years. M:F ratio was 2.3:1. Initial laboratory tests included routine investigations like complete blood count including red cell indices, peripheral smear evaluation, reticulocyte count, LDH levels, serum bilirubin levels, etc. Bone marrow examination was performed in select cases. Tests for identifying the cause of haemolytic anaemia and anaemia of ineffective erythropoiesis included Hb HPLC, incubated OFT, 5’ EMA dye binding test, methylene blue reduction test for G6PD screening, ARMS-PCR, GAP-PCR and electron microscopy. Cases diagnosed accurately with these initial set of tests were excluded from the study. Patients who remained undiagnosed and those with a phenotype of a haemolytic anaemia and a strong basis for inherited anaemia were included for further evaluation. Targeted next generation sequencing using panel of genes known to cause haemolytic anaemia was performed. The variants identified by NGS were confirmed by Sanger sequencing. Results: Twenty-three cases of anaemias were subjected to next generation sequencing. Twelve cases (n=12, 52%) showed the presence of a variant in the genes known to be associated with anaemia. The variants detected included those in genes involved in membrane proteins and channel proteins (SPTA, PIEZO1, ANK1, ABCG5), red cell enzymopathies (HK1, PKLR), beta-globin gene (HBB), CDA-II (SEC23-B), and other disorders (Primary hypertrophic osteoarthropathy and Autosomal visceral heterotaxy-8). (Figure-1). The variants were categorized as per ACMG guidelines and were either pathogenic or likely pathogenic. The disorders diagnosed included membranopathies & red cell channelopathies (hereditary spherocytosis, hereditary pyropoikilocytosis and sitosterolaemia), red cell enzymopathies (hexokinase deficiency, pyruvate kinase deficiency), β-thalassemia, congenital dyserythropoietic anaemia (CDA-II) and other syndromic disorders associated with secondary anemias (Primary hypertrophic osteoarthropathy and Autosomal visceral heterotaxy-8.Summary/Conclusion: Next generation sequencing methods are a valuable means to identify genetic basis of rare inherited anaemias. Routine tests for haemolytic anaemia may not diagnose all the cases. Red cell-based tests cannot be performed post blood transfusion. Using a limited gene panel, we were able to identify the genetic aetiology in 50% of the patients. Understanding the genetic basis helps better patient management and counselling. Antenatal genetic testing for future pregnancies can also be offered for the parents. Clinical exome sequencing can be planned in cases in which no variant is identified by targeted NGS. Keywords: Anemia, Genomics, Molecular